Apparatus and methods for magnetic mixing

ABSTRACT

Methods and systems for magnetic mixing. Particular embodiments relate to applying a magnetic field to move a magnetically responsive component in a chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/731,459, filed Jun. 5, 2015, which claims priority to U.S.Provisional Patent Application, Ser. No. 62/013,648 filed Jun. 18, 2014,the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

Embodiments of the present invention relate to mixing components usingmagnetic fields. Particular embodiments relate to applying a magneticfield to one or more chambers and moving a magnetically responsivecomponent in each chamber to mix contents in the chamber(s).

BACKGROUND

The following descriptions and examples are not admitted to be prior artby virtue of their inclusion within this section.

Chemical and biological reactions, including polymerase chain reactions(PCR), typically utilize multiple types of reagents in one or morephysical states. It is often desirable to mix the components of thereaction, for example to increase efficiency and/or consistency ofresults. Lyophilized reagents, in particular, may require more extensivemixing than liquids in order to rehydrate and distribute the reagents inthe reaction volume. In the context of PCR, for example, the reagentsare commonly mixed by pipetting the liquid up and down a number oftimes.

Mixing of components prior to or during PCR processes can presentseveral challenges. For example, it can be difficult to incorporate amixing mechanism in an enclosed sample-to-answer system. In addition, itmay be desirable to mix components in certain processes in a preciselycontrolled manner to reduce the likelihood of emulsification of thereaction components.

Exemplary embodiments of the present disclosure provide for mixing ofcomponents in a precisely controlled manner. In addition, exemplaryembodiments can be incorporated within the space limitations of PCRassemblies.

SUMMARY OF THE INVENTION

Exemplary embodiments of the present disclosure relate to systems andmethods for mixing contents in a chamber via a magnetic coupling betweencomponents located within and outside of the chamber.

Particular embodiments relate to applying a magnetic field to move amagnetically responsive component in the chamber. The magneticallyresponsive component may be, for example, a ball, disk, or rod. Inspecific embodiments, magnetic mixing using the magnetically responsivecomponent aids in one or more of: the displacement of air bubbles fromthe bottom or sides of the reaction volume, the resuspension oflyophilized reagents, and/or the inversion of wax that has not naturallyinverted by disrupting the surface tension at the wax-resuspensionbuffer interface. In certain embodiments, the magnetically responsivecomponent also disrupts the surface tension, which can allow for airbubbles to more easily escape from the liquid. Furthermore, magneticmixing may be used continuously or intermittently in order to reducetemperature gradients in the reaction volume. This can be advantageousin, for example, applications like PCR that involve heating and/orcooling of the chamber. In particular embodiments, during the mixingprocess, a magnet moves towards the chamber, which lifts themagnetically responsive component to just under the meniscus of theliquid, and then the magnet moves away from the chamber either by amotion that directs the magnetically responsive component to the bottomof the chamber by magnetic attraction or by a motion whereby themagnetically responsive component falls to the bottom of the chamber dueto gravity. In certain embodiments, the magnetically responsivecomponent is held at its upper position (e.g., near the meniscus) forabout 0, 1, 2, 3, 4, or 5 seconds, and is held at its lower position(e.g., at the bottom of the chamber) for about 0, 1, 2, 3, 4, or 5seconds. In particular embodiments, the movement of the magneticallyresponsive component between its upper and lower positions can becontinued for about 15, 30, 60, 90, 120, 180, 240 or 360 seconds, orbetween about 15 to 180 seconds, 30 to 120 seconds, or about 60 to 120seconds, or about 60 to 240 seconds, or about 60 to 360 seconds in orderto mix the contents of the chamber. In certain embodiments, the movementof the magnetically responsive component between its upper and lowerpositions is continuous or intermittent throughout the time of areaction occurring in the chamber. In particular embodiments, thechamber has a volume of between 25 μl and 2 ml, 25 μl and 1 ml, 25 μland 500 μl, 25 μl and 100 μl, 50 μl and 2 ml, 50 μl and 1 ml, or 50 μland 500 μl.

Certain embodiments include an apparatus comprising: a shaft comprisinga first end and a second end and a longitudinal axis extending betweenthe first end and the second end; a motor coupled to the shaft, wherethe motor is configured to rotate the shaft about the longitudinal axisof the shaft; a magnet coupled to the shaft, where the magnet comprisesa first end proximal to the longitudinal axis of the shaft and a secondend distal to the longitudinal axis of the shaft; and a housingconfigured to receive a chamber. In particular embodiments, the shaft isconfigured to move from a first shaft position to a second shaftposition, where, in the first shaft position, the second end of themagnet is distal from the housing, and in the second shaft position, thesecond end of the magnet is proximal to the housing.

In specific embodiments, the shaft is configured to rotate from thefirst shaft position to the second shaft position. In some embodiments,the housing comprises an insert configured to receive the chamber.Certain embodiments can further comprise a chamber received within thehousing, and a moveable magnetically responsive component disposedwithin the chamber. In particular embodiments, the moveable magneticallyresponsive component is in a first position when the shaft is in thefirst shaft position, and the moveable magnetically responsive componentis in a second position when the shaft is in the second shaft position.

In some embodiments, the moveable magnetically responsive component isin contact with the bottom surface of the chamber when the moveablemagnetically responsive component is in the first position, and themoveable magnetically responsive component is in contact with the sidesurface (and not the bottom surface) of the chamber when the moveablemagnetically responsive component is in the second position. In specificembodiments, the housing comprises a thermoelectric cooler (TEC). Incertain embodiments, the chamber comprises a composition of stabilizedlyophilized biological reagents. In particular embodiments, the sidesurface of the chamber is tapered and the bottom surface of the chamberis curved. In specific embodiments, the bottom surface is curved with afirst radius; the moveable magnetically responsive component is aspherical ball with a second radius; and the first radius is greaterthan the second radius. In certain embodiments, the second radius (i.e.,the radius of the ball) is at least 50%, 60%, 70%, 80%, or 90% (but lessthan 100%) of the first radius (i.e., the radius of the bottom surface).

In particular embodiments, the moveable magnetically responsivecomponent is a ball, a disk, or a rod. In certain aspects, themagnetically responsive component may have a diameter or length in itslongest dimension of between about 0.5 mm to about 5 mm, or betweenabout 1 mm to about 2 mm. In some embodiments, the rod has a lengthbetween approximately 0.0625 inches and 0.125 inches. In specificembodiments, the moveable magnetically responsive component is astainless steel ball. In particular embodiments, the stainless steelball is a 400 series stainless steel ball. In some embodiments the ballhas a diameter of about 1.6 mm. In some embodiments, the stainless steelobject (e.g., ball, disk, or rod) has been passivated to remove freeiron or other inclusions from its surface. Stainless steel can bepassivated by, for example, a series of acid baths, which clean freeiron or other inclusions from the surface, and form a uniform naturaloxide layer that protects the stainless steel from corrosion. Themagnetically responsive component is preferably made of, or at leastcoated with, a material that is inert to the reaction conditions inwhich it is present. In certain aspects, magnetic or magneticallyresponsive materials may be encased in or coated with non-magneticallyresponsive material in order to prevent their interaction with thereaction environment. For example, the magnetic or magneticallyresponsive materials may be encased in or coated with ceramic, glass, orplastic (e.g., polystyrene, polyethylene, polyethene, polypropylene,neoprene, poly(tetrafluoroethylene)).

In certain embodiments, the chamber comprises contents suitable for usein a polymerase chain reaction (PCR) nucleic acid amplification processand the moveable magnetically responsive component is passivated to forman oxide layer that is non-reactive with contents of the chamber. Inparticular embodiments, the chamber comprises reagents suitable for usein polymerase chain reaction (PCR) nucleic acid amplification process.

In some embodiments, the first shaft position is approximately, 15, 20,25, 30, 35, 50, 60, 70, 80, or 90 degrees from the second shaftposition. In some embodiments, the first shaft position is between about15 to 90 degrees, 20 to 50 degrees, or 20 to 30 degrees from the secondshaft position. Specific embodiments further comprise a switchconfigured to limit rotation of the shaft between the first shaftposition and the second shaft position. In certain embodiments, theswitch is an optical switch comprising a disc coupled to the shaft. Inparticular embodiments, the apparatus is coupled to a polymerase chainreaction (PCR) control module configured to control rotation of theshaft between the first shaft position and the second shaft position. Insome embodiments, the PCR control module is configured to controlrotation of the shaft from the first shaft position to the second shaftposition such that the moveable magnetically responsive component isheld in the first position for approximately 0, 1, 2, 3, 4, 5 seconds(or any range therein) and then moved to the second position and held inthe second position for approximately 0, 1, 2, 3, 4, 5 seconds (or anyrange therein). In specific embodiments, the PCR control module isconfigured to control rotation of the shaft from the first shaftposition to the second shaft position such that the moveablemagnetically responsive component is cycled between the first and secondpositions for approximately 15, 30, 60, 90, 120, 180, 240 or 360 seconds(or any range therein). In some embodiments, the PCR control module isconfigured to control rotation of the shaft from the first shaftposition to the second shaft position such that the moveablemagnetically responsive component is cycled between the first and secondpositions prior to concurrent with the start of the PCR (e.g., during aninitial denaturation step or during a reverse transcription phase if itis an RT-PCR), intermittently during the PCR (e.g., mixing duringtemperature changes to reduce thermal gradients in the reaction), orcontinuously during all or a substantial portion of the PCR.

In certain embodiments, the magnet has a maximum energy product (BHmax)of between 48 and 54 Megagauss-Oersteds (MGOe). In some embodiments, themagnet has a maximum energy product (BHmax) of approximately 52 MGOe. Inparticular embodiments, the second end of the magnet is locatedapproximately 0.70, 0.75, 0.80, 0.82, 0.85, or 0.90 (or any rangetherein) inches from the longitudinal axis of the rotating shaft. Insome embodiments, the second end of the magnet is approximately 0.10,0.15, 0.20, 0.21, 0.22, or 0.25 (or any range therein) inches from acentroid of the moveable magnetically responsive component when thesystem is in the first shaft position. The apparatus may be configuredsuch that the magnet is only close enough to magnetically attract themagnetically responsive component when the magnet is in the secondposition or it may be configured such that the magnet is close enough tomagnetically attract the magnetically responsive component when themagnet is in both the second and first positions. Magneticallyattracting the magnetically responsive component to its lower positionprovides the ability, if desired, to move the magnetically responsivecomponent to the lower position faster than with gravity alone. Inspecific embodiments, the magnet is longitudinally magnetized. Incertain embodiments, the magnet is a made of neodymium. In particularembodiments, the magnet is cylindrical in shape, with a diameter ofapproximately 0.125 inches and a length of 0.375 inches. In someembodiments, the housing comprises a first opening configured to receivethe magnet such that the second end of the magnet extends into the firstopening when the shaft is in the second shaft position. In certainembodiments, the second end of the magnet also may extend into orpartially into the first opening when the shaft is in the first shaftposition. In specific embodiments, the housing comprises a secondopening configured to receive a chamber. In particular embodiments, thehousing comprises a third opening configured to receive a fiber-opticcable. In some embodiments, the housing comprises a fourth openingconfigured to receive a second fiber-optic cable. In certainembodiments, the housing comprises an insert defining a conical space.

Some embodiments include an apparatus comprising: a shaft comprising afirst end and a second end and a longitudinal axis extending between thefirst end and the second end; a motor coupled to the shaft, wherein themotor is configured to rotate the shaft about the longitudinal axis ofthe shaft; a plurality of magnets coupled to the shaft along thelongitudinal axis of the shaft, wherein each magnet comprises a firstend proximal to the longitudinal axis of the shaft and a second enddistal to the longitudinal axis of the shaft; and a module comprising aplurality of housings arranged along a linear axis. The module maycomprise, for example, 4 to 24 housings, 4 to 12 housings, 4 to 8housings, 4 to 6 housings, 6 to 24 housings, or 6 to 12 housings. Inspecific embodiments, the longitudinal axis of the shaft issubstantially parallel to the linear axis of the plurality of housings;and each magnet of the plurality of magnets is aligned with acorresponding housing of the plurality of housings.

In certain embodiments, the shaft is configured to rotate from a firstshaft position in which the second end of each magnet is distal from thecorresponding housing; and the shaft is configured to rotate to a secondshaft position in which the second end of each magnet is proximal fromthe corresponding housing. Particular embodiments further comprise: achamber disposed within each housing of the plurality of housings; and amoveable magnetically responsive component disposed within each chamber.In some embodiments, the second end of each magnet is distal to a sidesurface of the chamber when the shaft is in the first position; and thesecond end of each magnet is proximal to a side surface of the chamberand distal to the bottom surface of the chamber when the shaft is in thesecond position. In specific embodiments, the moveable magneticallyresponsive component in each chamber is in a first position in contactwith a bottom surface of the chamber when the shaft is in the firstshaft position; and the moveable magnetically responsive component ineach chamber is in a second position in contact with a side surface ofthe chamber and not in contact with a bottom surface of the chamber whenthe shaft is in the second shaft position.

In certain embodiments, the side surface of each chamber is tapered andthe bottom surface of each chamber is curved. In particular embodiments,the housing comprises a thermoelectric cooler (TEC). In someembodiments, each chamber comprises a composition of stabilizedlyophilized biological reagents comprising a lyophilized pelletcomprising nucleoside triphosphates (NTPs) and a polymerase enzyme. Inparticular embodiments, the moveable magnetically responsive componentis a stainless steel ball. In certain aspects, the magneticallyresponsive component may have a diameter or length in its longestdimension of between about 0.5 mm to about 5 mm, or between about 1 mmto about 2 mm. In some embodiments, the moveable magnetically responsivecomponent is a sphere (i.e., ball) with a diameter of approximately0.0625 inches. In specific embodiments, each chamber comprises contentssuitable for use in a polymerase chain reaction (PCR) nucleic acidamplification process and wherein the moveable magnetically responsivecomponent is passivated to form an oxide layer that is non-reactive withcontents of the chamber. In certain embodiments, each chamber comprisesreagents suitable for use in polymerase chain reaction (PCR) nucleicacid amplification process. In particular embodiments, each chamberfurther comprises a liquid.

In some embodiments, the moveable magnetically responsive component ineach chamber is in a first position in contact with a bottom surface ofthe chamber when the shaft is in the first shaft position; the moveablemagnetically responsive component in each chamber is in a secondposition that contacts a side surface of the chamber when the shaft isin the second shaft position; and the second position of the moveablemagnetically responsive component is located between the surface of theliquid and the bottom surface of the chamber. In specific embodiments,the first shaft position is approximately 15, 20, 25, 30, 35, 50, 60,70, 80, or 90 degrees from the second shaft position. In someembodiments, the first shaft position is between about 15 to 90 degrees,20 to 50 degrees, or 20 to 30 degrees from the second shaft position.

Certain embodiments further comprise a switch configured to limitrotation of the shaft between the first shaft position and the secondshaft position. In particular embodiments, the switch is an opticalswitch comprising a disc coupled to the shaft. In some embodiments, theapparatus is coupled to a polymerase chain reaction (PCR) control moduleconfigured to control rotation of the shaft between the first shaftposition and the second shaft position. In specific embodiments, the PCRcontrol module is configured to control rotation of the shaft from thefirst shaft position to the second shaft position such that the moveablemagnetically responsive component is held in the first position forapproximately 0, 1, 2, 3, 4, 5 seconds (or any range therein) and thenmoved to the second position and held in the second position forapproximately 0, 1, 2, 3, 4, 5 seconds (or any range therein). Incertain embodiments, the PCR control module is configured to controlrotation of the shaft from the first shaft position to the second shaftposition such that the moveable magnetically responsive component iscycled between the first and second positions for approximately 15, 30,60, 90, 120, 180, 240 or 360 seconds (or any range therein). In someembodiments, the PCR control module is configured to control rotation ofthe shaft from the first shaft position to the second shaft positionsuch that the moveable magnetically responsive component is cycledbetween the first and second positions prior to concurrent with thestart of the PCR (e.g., during an initial denaturation step or during areverse transcription phase if it is an RT-PCR), intermittently duringthe PCR (e.g., mixing during temperature changes to reduce thermalgradients in the reaction), or continuously during all or a substantialportion of the PCR.

Other embodiments include methods of mixing reagents using an apparatusas disclosed herein. The method may comprise, for example: obtaining anapparatus comprising: a chamber containing a magnetically responsivecomponent and reagents; and a magnet coupled to a rotating shaft, where:the shaft is configured to move from a first shaft position to a secondshaft position; in the first shaft position, the second end of themagnet is distal from the chamber; and in the second shaft position, thesecond end of the magnet is proximal to the chamber; and moving theshaft from a first position to a second position, wherein themagnetically responsive component is moved within the chamber from afirst position to a second position, thereby mixing the reagents. Thereagents may be, for example, PCR reagents and/or reverse transcriptionreagents. In some embodiments, the reagents may comprise an enzyme. Incertain embodiments, the enzyme may be a polymerase, an endonuclease, oran exonuclease. Certain embodiments include a method of mixing reagentsduring a polymerase chain reaction (PCR). In specific embodiments themethod includes obtaining an apparatus comprising: a chamber containinga magnetically responsive component and reagents, wherein the reagentsare suitable for PCR; and a magnet configured to move the magneticallyresponsive component between a first position in the chamber and asecond position in the chamber (e.g. between a bottom surface of thechamber and a side surface of the chamber. Particular embodiments caninclude a magnet coupled to a rotating shaft, where the shaft isconfigured to move from a first shaft position to a second shaftposition, and in the first shaft position, the second end of the magnetis distal from the chamber, and in the second shaft position, the secondend of the magnet is proximal to the chamber. In certain embodiments,the method includes moving the shaft from a first position to a secondposition, wherein the magnetically responsive component is moved withinthe chamber from a first position to a second position, thereby mixingthe reagents.

In particular embodiments, the chamber comprises a bottom surface and aside surface, and wherein the magnetically responsive component contactsthe bottom surface in the first position and wherein the magneticallyresponsive component contacts the side surface in the second position.In some embodiments, the magnetically responsive component is moved fromthe first position to the second position and held in the secondposition for approximately 0, 1, 2, 3, 4, 5 seconds (or any rangetherein); and the magnetically responsive component is moved from thesecond position to the first position and held in the first position forapproximately 0, 1, 2, 3, 4, 5 seconds (or any range therein). Inspecific embodiments, the magnetically responsive component is cycledbetween the first and second positions for approximately 15, 30, 60, 90,120, 180, 240 or 360 seconds (or any range therein). In certainembodiments, the moveable magnetically responsive component is cycledbetween the first and second positions prior to or concurrent with thestart of the PCR (e.g., during an initial denaturation step or during areverse transcription phase if it is an RT-PCR), intermittently duringthe PCR (e.g., mixing during temperature changes to reduce thermalgradients in the reaction), or continuously during all or a substantialportion of the PCR. In particular embodiments, at least one of thereagents is provided in a lyophilized form. In some embodiments, theside surface of the chamber is tapered and the bottom surface of thechamber is curved. In specific embodiments of the method, the bottomsurface is curved with a first radius; the moveable magneticallyresponsive component is a spherical ball with a second radius; and thefirst radius is greater than the second radius. Certain embodiments ofthe method comprise moving the magnetically responsive component priorto beginning a first PCR cycle. Particular embodiments comprise movingthe magnetically responsive component during at least a portion of eachPCR cycle.

In some embodiments, the movement of the magnetically responsivecomponent occurs during a temperature ramping phase. In specificembodiments, movement of the magnetically responsive component from thefirst position to the second position reduces a temperature gradient inthe chamber. Certain embodiments comprise moving the magneticallyresponsive component prior to and during a polymerase chain reaction.Particular embodiments comprise moving the magnetically responsivecomponent prior to beginning a reverse transcription reaction. Someembodiments comprise moving the magnetically responsive component duringat least a portion of a reverse transcription reaction. Specificembodiments comprise moving the magnetically responsive component priorto and during a polymerase chain reaction.

In certain embodiments, movement of the magnetically responsivecomponent from the first position to the second position inverts amelted wax layer in the chamber. In particular embodiments of themethods, the magnetically responsive component is a sphere. In someembodiments of the method, the sphere has a diameter of approximately0.0625 inches. In specific embodiments of the method, the magneticallyresponsive component is a disk or a sphere having a first diameter, andwherein a distance from first position to the second position of themagnetically responsive component is between two and five times thefirst diameter. In certain embodiments, the reagents are polymerasechain reaction (PCR) reagent. In particular embodiments, the reagentsare reverse transcription reagents. In some embodiments, the reagentscomprise an enzyme, and in specific embodiments, the enzyme is apolymerase, endonuclease, or a exonuclease.

Certain embodiments include an apparatus comprising: a housing; aninsert disposed within the housing, wherein the insert is configured toreceive a chamber; a first electromagnet proximal to a first location onthe insert; and a second electromagnet proximal to a second location onthe insert. In particular embodiments, the first and secondelectromagnets are configured to alternatingly and respectively apply amagnetic force to the first and second locations on the insert. In someembodiments, the insert comprises a conical space defined by a taperedside surface having a first end and a second end; the first end islarger in diameter than the second end; the first end is open and thesecond end is closed; the first location on the insert is locatedbetween the first end and the second end of the insert; and the secondlocation is located proximal to the second end of the insert.

Some embodiments further comprise: a chamber received within the insert;and a moveable magnetically responsive component disposed within thechamber. In specific embodiments, the moveable magnetically responsivecomponent is in a first position when the first electromagnet isactivated to apply a magnetic force; and the moveable magneticallyresponsive component is in a second position when the secondelectromagnet is activated to apply a magnetic force. In certainembodiments, the chamber comprises contents suitable for use in apolymerase chain reaction (PCR) nucleic acid amplification process andthe moveable magnetically responsive component is passivated to form anoxide layer that is non-reactive with contents of the chamber. Inparticular embodiments, the chamber comprises reagents suitable for usein polymerase chain reaction (PCR) nucleic acid amplification process.

Certain embodiments include an apparatus comprising: a housing; aninsert disposed within the housing, wherein the insert is configured toreceive a chamber; and an electromagnet proximal to a first location onthe insert, where the electromagnet is configured to alternatingly applya magnetic force to the location on the insert. In specific embodiments,the insert comprises a conical space defined by a tapered side surfacehaving a first end and a second end; the first end is larger in diameterthan the second end; the first end is open and the second end is closed;and the first location on the insert is located between the first endand the second end of the insert.

Certain embodiments further comprise: a chamber received within theinsert; and a moveable magnetically responsive component disposed withinthe chamber. In particular embodiments, the moveable magneticallyresponsive component is in a first position when the electromagnet isenergized to apply a magnetic force; and the moveable magneticallyresponsive component is in a second position when the electromagnet isnot energized to apply a magnetic force. In certain embodiments, thechamber comprises contents suitable for use in a polymerase chainreaction (PCR) nucleic acid amplification process and the moveablemagnetically responsive component is passivated to form an oxide layerthat is non-reactive with contents of the chamber. In particularembodiments, the chamber comprises reagents suitable for use inpolymerase chain reaction (PCR) nucleic acid amplification process.

The term “coupled” is defined as connected, although not necessarilydirectly, and not necessarily mechanically. Two items are “coupleable”if they can be coupled to each other, and, when coupled, may still becharacterized as “coupleable.” Unless the context explicitly requiresotherwise, items that are coupleable are also decoupleable, andvice-versa. One non-limiting way in which a first structure iscoupleable to a second structure is for the first structure to beconfigured to be coupled (or configured to be coupleable) to the secondstructure.

The terms “a” and “an” are defined as one or more unless this disclosureexplicitly requires otherwise.

The term “substantially” and its variations (e.g., “approximately” and“about”) are defined as being largely but not necessarily wholly what isspecified (and include wholly what is specified) as understood by one ofordinary skill in the art. In any disclosed embodiment, the terms“substantially,” “approximately,” and “about” may be substituted with“within [a percentage] of” what is specified, where the percentageincludes 0.1, 1, 5, and 10 percent.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “include” (and any form of include, such as “includes” and“including”) and “contain” (and any form of contain, such as “contains”and “containing”) are open-ended linking verbs. As a result, a method ordevice that “comprises,” “has,” “includes” or “contains” one or moresteps or elements possesses those one or more steps or elements, but isnot limited to possessing only those one or more elements. Likewise, astep of a method or an element of a device that “comprises,” “has,”“includes” or “contains” one or more features possesses those one ormore features, but is not limited to possessing only those one or morefeatures. For example, a system that comprises an ultrasonic transducerhas one sample reservoir unit, but may have more than one ultrasonictransducer.

Furthermore, a device or structure that is configured in a certain wayis configured in at least that way, but may also be configured in waysthat are not listed. Metric units may be derived from the English unitsprovided by applying a conversion and rounding to the nearestmillimeter.

The feature or features of one embodiment may be applied to otherembodiments, even though not described or illustrated, unless expresslyprohibited by this disclosure or the nature of the embodiments.

Any embodiment of any of the disclosed devices and methods can consistof or consist essentially of—rather thancomprise/include/contain/have—any of the described elements and/orfeatures and/or steps. Thus, in any of the claims, the term “consistingof” or “consisting essentially of” can be substituted for any of theopen-ended linking verbs recited above, in order to change the scope ofa given claim from what it would otherwise be using the open-endedlinking verb.

Other features and associated advantages will become apparent withreference to the following detailed description of specific embodimentsin connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings illustrate by way of example and not limitation.For the sake of brevity and clarity, every feature of a given structuremay not be labeled in every figure in which that structure appears.Identical reference numbers do not necessarily indicate an identicalstructure. Rather, the same reference number may be used to indicate asimilar feature or a feature with similar functionality, as maynon-identical reference numbers.

FIG. 1 is perspective view of a polymerase chain reaction (PCR) modularassembly comprising a magnetic actuation assembly and thermo-electriccooler (TEC) sub-assembly according to exemplary embodiments of thepresent disclosure.

FIG. 2 is a perspective view of the magnetic actuation assembly of theembodiment of FIG. 1.

FIG. 3 is a perspective view of the (TEC) sub-assembly of the embodimentof FIG. 1.

FIG. 4 is a first perspective view of the magnetic actuation assemblyand the (TEC) sub-assembly of the embodiment of FIG. 1.

FIG. 5 is a second perspective view of a partial magnetic actuationassembly and the (TEC) sub-assembly of the embodiment of FIG. 1.

FIG. 6 is a third perspective view of a partial magnetic actuationassembly and the (TEC) sub-assembly of the embodiment of FIG. 1.

FIG. 7 is a partial section view of the magnetic actuation assembly andthe (TEC) sub-assembly of the embodiment of FIG. 1 in a first position.

FIG. 8 is a partial section view of the magnetic actuation assembly andthe (TEC) sub-assembly of the embodiment of FIG. 1 in a second position.

FIG. 9 is a partial perspective view of the magnetic actuation assemblyand the (TEC) sub-assembly of the embodiment of FIG. 1 in a firstposition.

FIG. 10 is a partial perspective view of the magnetic actuation assemblyand the (TEC) sub-assembly of the embodiment of FIG. 1 in a secondposition.

FIG. 11 is a graph of relative fluorescence units (RFU) detected duringPCR plotted against PCR cycles performed by the embodiment of FIG. 1.

FIG. 12 is a graph of the delta relative fluorescence units (RFU)detected during melt plotted against temperature performed by theembodiment of FIG. 1.

FIG. 13 is a perspective view of a partial magnetic actuation assemblyand a (TEC) sub-assembly.

FIG. 14 is a partial section view of the embodiment of FIG. 13.

FIG. 15 is a partial section view of a PCR modular assembly comprisingelectromagnets.

FIG. 16 is a partial section view of a PCR modular assembly comprising asingle electromagnet.

DETAILED DESCRIPTION

Various features and advantageous details are explained more fully withreference to the non-limiting embodiments that are illustrated in theaccompanying drawings and detailed in the following description. Itshould be understood, however, that the detailed description and thespecific examples, while indicating embodiments of the invention, aregiven by way of illustration only, and not by way of limitation. Varioussubstitutions, modifications, additions, and/or rearrangements willbecome apparent to those of ordinary skill in the art from thisdisclosure.

In the following description, numerous specific details are provided toprovide a thorough understanding of the disclosed embodiments. One ofordinary skill in the relevant art will recognize, however, that theinvention may be practiced without one or more of the specific details,or with other methods, components, materials, and so forth. In otherinstances, well-known structures, materials, or operations are not shownor described in detail to avoid obscuring aspects of the invention. Itis understood that for purposes of clarity, not all reference numbersare shown for every component visible in each figure.

Referring initially to FIGS. 1-6, a polymerase chain reaction (PCR)modular assembly 50 configured for nucleic acid amplification comprisesa magnetic actuation assembly 100 coupled to a thermo-electric cooler(TEC) sub-assembly 200. In the embodiment shown, (PCR) modular assembly50 also comprises a PCR control module 60 configured to control magneticactuation assembly 100, including for example, the movement or rotationof a shaft 110 of magnetic actuation assembly 100.

Referring particularly now to FIGS. 2-6, magnetic actuation assembly 100comprises shaft 110 coupled to an electric motor 120 via a coupling 125(e.g., a bellows coupling). In the embodiment shown, magnetic actuationassembly 100 also comprises a support plate 130 and support members 135that support shaft 110. In particular embodiments, support members 135can be configured as pillow blocks containing plastic bushings. Magneticactuation assembly 100 may also comprise one or more switches 115 thatcan limit rotation of shaft 110, as explained in further detail below.

In the embodiment shown, shaft 110 comprises a first end 111 and asecond end 112 with a longitudinal axis 113 extending between the firstand second end. The embodiment shown also comprises a plurality ofmagnets 150 coupled to shaft 110 along longitudinal axis 113.

Magnetic actuation assembly 100 also comprises a plurality of retentionmembers 160 configured to retain other components (e.g. fiber-opticcables 161) from interfering with operation of magnetic actuationassembly 100. As shown in FIGS. 4 and 5, TEC sub-assembly 200 can alsocomprise a heating module 201 and a heat sink 202.

As shown in FIGS. 3 and 6, TEC sub-assembly 200 comprises a plurality ofinserts 250 each disposed within a housing 205. In exemplaryembodiments, inserts 250 can be configured as aluminum TEC blocksconfigured to conduct thermal energy to a chamber 230 (e.g. a PCR tube)as shown in FIGS. 7 and 8. Housing 205 may be configured as a TEC blockinsulator in exemplary embodiments.

As shown in FIGS. 7 and 8, an insert 250 can be configured to receive achamber 230. In particular embodiments, insert 250 comprises a conicalspace 210 configured to receive chamber 230. In the embodiment shown,conical space 210 of insert 250 is defined by a tapered side surface 270having a first end 271 that is larger and open, and a second end 272that is smaller and closed. In the embodiment shown, the plurality ofhousings 205 are arranged along a linear axis 217 that is substantiallyparallel to longitudinal axis 113 of shaft 110 (shown in FIG. 5). In theillustrated embodiment each magnet 150 is aligned with a correspondinghousing 205.

In particular embodiments, each chamber 230 comprises a side surface 211that is tapered and a bottom surface 212 that is curved, such that sidesurface 211 and bottom surface 212 form a generally conical structure.It is understood that the terms “side surface” and “bottom surface” usedthroughout this disclosure are used only for reference purposes withrespect to the drawings. For example, bottom surface 212 does notnecessarily have to be at an absolute lowest portion of chamber 230,depending on the orientation of chamber 230. During operation, chambers230 may comprise many different components used for PCR nucleic acidamplification. For example chambers 230 may comprise reagents includingbuffers, nucleotides, modified nucleotides, primers, probes, enzymes,sugars, and stabilizers.

In certain instances, it can be desirable to ensure the reagents aresufficiently mixed together to promote efficiency and accuracy in thePCR process. However, mixing of the components can also createundesirable effects. For example, mixing can create bubbles thatinterfere with the optical detection by fiber-optic cables 161. Inaddition, certain PCR processes can include an insulating layer 213(e.g. an oil or wax layer) on top of the reagents 214 to reduceevaporation. In specific embodiments, insulating layer 213 may comprise25 of docosane wax or mineral oil. If the mixing process is not properlycontrolled, insulating layer 213 can become emulsified with reagents214, thereby increasing evaporation and reducing accuracy in the PCRdetection and analysis.

Embodiments of the present disclosure provide for mixing of the PCRreagents in a controlled manner that reduces the likelihood of unwantedbubble formation or emulsification of insulating layer 213 and reagents214. Particular embodiments comprise a moveable magnetically responsivecomponent 220 disposed within a chamber 230. In certain embodiments,moveable magnetically responsive component 220 may be configured as amagnetic 400 series stainless steel (e.g. 440C grade) ball that ispassivated to form a non-reactive oxide layer. In specific embodiments,moveable magnetically responsive component 220 may be sized in relationto the dimensions of bottom surface 212 of chamber 230. For example,moveable magnetically responsive component 220 can be a magnetic ballsized to engage the lowest portion of bottom surface 212 (e.g. theportion distal from insulating layer 213) without simultaneouslyengaging tapered side surface 211. In particular, moveable magneticallyresponsive component 220 can be a spherical or ball shape with a radiusR1 that is less than a radius R2 of bottom surface 212. This can allowmoveable magnetically responsive component 220 to adequately engage andmix the contents throughout chamber 230 without trapping bubbles betweenmoveable magnetically responsive component 220 and bottom surface 212.In specific embodiments, moveable magnetically responsive component 220can be configured as a spherical ball having a 1/16 (0.0625) inchdiameter (i.e. a 1/32 or 0.03125 inch radius).

Referring now to FIGS. 7-10, magnetic actuation assembly 100 can beactuated such that shaft 110 is moved (e.g. rotated) from a first shaftposition 117 to a second shaft position 119. As shown in FIGS. 7 and 8,magnet 150 is coupled to shaft 110 via a coupler 155 that spaces magnet150 away from axis 113 of shaft 110. In this embodiment, each magnet 150comprises a first end 151 proximal to longitudinal axis 113 and a secondend 152 distal to longitudinal axis 113. Such a configuration allowssecond end 152 to swing in a wider rotational arc than first end 151 asshaft 110 is rotated.

In the embodiment shown, switch 115 (shown in FIG. 2) can limit rotationof shaft 110 between first shaft position 117 and second shaft position119. In particular embodiments, switch 115 may be configured as anoptical switch that limits rotation of shaft 110 to approximately 25degrees between the first shaft position and the second shaft position.In specific embodiments, switch 115 may comprise a disc 116 that breaksan optical path within switch 115 to control rotation of shaft 110.

In first shaft position 117 (shown in FIGS. 7 and 9) second end 152 ofmagnet 150 is distal from chamber 230. In the first position, moveablemagnetically responsive component 220 also contacts bottom surface 212of chamber 230. In particular embodiments, magnet 150 may be anaxially-magnetized magnet. Such a configuration can allow magnet 150 toexert a magnetic force on moveable magnetically responsive component 220towards bottom surface 212 when shaft 110 is in the first position. Thiscan help overcome viscosity drag forces between moveable magneticallyresponsive component 220 and reagents 214 and assist moveablemagnetically responsive component 220 to contact bottom surface 212. Incertain cases, the force of gravity alone may not be sufficient toovercome the viscous forces to ensure contact between moveablemagnetically responsive component 220 and bottom surface 212.

In second shaft position 119 of shaft 110 (shown in FIGS. 8 and 10),second end 152 of magnet 150 is proximal to chamber 230 and moveablemagnetically responsive component 220 contacts side surface 211 ofchamber 230 as a result of the magnetic force exerted by magnet 150. Asshown in FIG. 8, in second shaft position 119, moveable magneticallyresponsive component 220 is located below an interface 215 of insulatinglayer 213 and reagents 214 (e.g. between interface 215 and bottomsurface 212). The relocation of moveable magnetically responsivecomponent 220 between the first position contacting bottom surface 212and the second position contacting side surface 211 can promote mixingof the contents of chamber 230.

As shown in FIGS. 3, 9 and 10, each housing 205 may include a slot oropening 206 facing a magnet 150. In the first position shown in FIG. 9,magnet 150 is proximal to the lower portion of opening 206, and in thesecond position shown in FIG. 10, magnet 150 extends into opening 206and is proximal to the upper end of opening 206. Opening 206 isconfigured to receive magnet 150 such that second end 152 of magnet 150extends into opening 206 when shaft 110 is in second shaft position 119.

In certain embodiments, housing 205 may function as an insulator or heatblock to retain thermal energy in chamber 230 provided by heating module201. In addition, housing 205 may comprise openings 207 for receivingand coupling fiber-optic cables 161. Furthermore, housing 205 maycomprise an opening 208 for receiving chamber 230 and tapered wall 221(defining a generally conical shape) configured to engage side surface270 of chamber 230.

In particular embodiments, moveable magnetically responsive component220 can be held in the second position for approximately 3 seconds, andthen moved back to the first position for approximately 3 seconds to mixthe contents of chamber 230. In certain embodiments, this cycling ofmoveable magnetically responsive component 220 between the first andsecond positions can be repeated for approximately 90 seconds. Inparticular embodiments, the rotation of shaft 110 between the firstshaft position and the second shaft position can be controlled by PCRcontrol module 60 of PCR modular assembly 50.

In specific embodiments, chamber 230 may comprise biological reagentsthat are inherently unstable at ambient temperatures and are stabilizedwith sugars via lyophilization. Lyophilization of biological reagentsresults in generation of material with low moisture content (e.g., lessthan 5 percent) and the functionality of the lyophilized material iscompromised if it is not stored dry. Continued stability of lyophilizedmaterial therefore requires methods to prevent moisture absorption whichincludes secondary containers, storage in dry humidity environment, etc.In certain examples, a layer of wax can be used to create a moisturebarrier for the lyophilized material that improves the stability oflyophilized reagents.

In certain embodiments, lyophilized material can be stabilized withinsulating layer 213, which allows for storage of sample extractioncassette at ambient conditions without special requirements for a lowhumidity environment. As previously mentioned, insulating layer 213 canalso used as a vapor barrier during PCR to reduce or preventevaporation. After PCR cycling, insulating layer 213 (e.g. wax) can alsosolidify and create a full or partial barrier to potential ampliconcontamination. An amplicon can be difficult to eliminate if itcontaminates a lab and the solid wax significantly reduces the chance ofsuch an occurrence.

The mixing process described herein can assist in the inversion ofinsulating layer 213 that has not naturally inverted by disrupting thesurface tension at the insulating layer-resuspension buffer interface.Moveable magnetically responsive component 220 can also disrupt thesurface tension, allowing for air bubbles that may be caught in theresuspension buffer to be released and rise to the top. Furthermore, themagnetic mixing process described herein can be used to mix theresuspension buffer with the lyophilized cake and promote uniformdistribution of components, as well as reduce a temperature gradientwithin chamber 230.

Examples of the benefits of mixing contents of chamber 230 can beillustrated in FIGS. 11 and 12. In FIG. 11, the relative fluorescenceunits (RFU) detected during PCR are plotted against PCR cycles. FIG. 12illustrates the delta in RFU plotted against temperature in a derivativemelt curve. In FIGS. 11 and 12 the lighter/dotted line illustratesresults from of contents that are not mixed in the PCR chamber, whilethe darker (non-dotted) lines illustrated results from contents that aremixed. FIG. 11 shows that the non-mixed results did not reach thedesired baseline RFU value until approximately 18-20 cycles had beenperformed and there was a delay in shoulder between mixed and non-mixed.FIG. 12 shows the delta RFU is substantially reduced in the non-mixedresults as compared to those of the mixed results.

Other exemplary embodiments may utilize different components orconfigurations from those disclosed above. For example, certainexemplary embodiments may comprise a rotating magnetic rod rather than aplurality of magnets coupled to a rotating rod. Referring now to FIGS.13-14, TEC 200 is coupled to a magnetic actuation assembly 400 thatcomprises a rotating magnetic rod 450 supported by brackets 460 and 470.As shown in the axial view of FIG. 14, magnetic rod 450 is radiallymagnetized such that the north pole (N) of the magnetic field extendsfrom location on the circumference of the rod and the south pole (S) ofthe magnetic field extends from a location approximately 180 degreescircumferentially from the north pole. Accordingly, as magnetic rod 450rotates along axis 413, the north and south poles N and S will bedirected toward insert 250 and a PCR chamber (not shown) inserted intoinsert 250. Similar to previously described embodiments, the alternatingmagnetic field can direct movement of a moveable magnetically responsivecomponent contained within a PCR chamber disposed within insert 250.Such movement can be used for multiple purposes, including for example,mixing components or reducing a temperature gradient.

In addition to the previously described embodiments, certain embodimentsmay utilize electromagnets to apply a magnetic force to the contents ofa PCR chamber, including a moveable magnetically responsive component.Referring now to FIG. 15, a polymerase chain reaction (PCR) modularassembly 40 configured for nucleic acid amplification comprises a firstelectromagnet 351 and a second electromagnet 352. Similar to previousembodiments, this embodiment also comprises housing 205 and insert 250.For purposes of clarity, not all features of insert 250 are labeled inFIG. 15, but it is understood that insert 250 in FIG. 15 comprisesfeatures equivalent to those shown in FIGS. 7 and 8 (including forexample, tapered side surface 270 having first end 271 that is largerand open, and second end 272 that is smaller and closed.)

In this embodiment, first electromagnet 351 is proximal to a firstlocation 261 on insert 250 that is located between first end 271 andsecond end 272. Second electromagnet 352 is proximal to a secondlocation 262 that is proximal to second end 272 of insert 250. First andsecond electromagnets 351 and 352 are configured to alternatingly andrespectively apply a magnetic force to first and second locations 261and 262 on insert 250. For example, first electromagnet 351 can beenergized to apply a magnetic force to first location 261 while secondelectromagnet 352 is not energized to exert a magnetic force.Subsequently, second electromagnet 352 can be energized to apply amagnetic force to second location 352 while first electromagnet 352 isnot energized to apply a magnetic force. This pattern can be repeatedsuch that magnetic forces are alternatingly applied to first and secondlocations 261 and 262.

Accordingly, as first and second electromagnets 351 and 352 arealternatingly energized to apply magnetic forces to first and secondlocations 261 and 262, respectively, the magnetic field will be variedwithin insert 250 and a PCR chamber (not shown) inserted into insert250. Similar to previously described embodiments, the alternatingmagnetic field can direct movement of a moveable magnetically responsivecomponent contained within a PCR chamber disposed within insert 250.Such movement can be used for multiple purposes, including for example,to mix components or reduce a temperature within the insert or a chamberdisposed within the insert.

Referring now to FIG. 16, another exemplary embodiment comprises asingle electromagnet 551. This embodiment is similar to the embodimentdescribed in FIG. 15, but allows the force of gravity to directmagnetically responsive component 220 to bottom surface 212 of chamber230 (instead of a magnetic force applied by a second electromagnet). Inthis embodiment, electromagnet 551 can be energized to apply a magneticforce and direct magnetically responsive component 220 to side surface211 of chamber 230. Electromagnet 551 can then be de-energized to reduceor eliminate the magnetic force applied to magnetically responsivecomponent 220, allowing magnetically responsive component 220 to fall tobottom surface 212 of chamber 230. Electromagnet 551 can bealternatingly energized and de-energized to move the ball from a firstlocation (e.g. side surface 211) to a second location (e.g. bottomsurface 212). Such movement can be used, for example, to mix componentsor reduce a temperature within the insert or a chamber disposed withinthe insert.

Still other embodiments may comprise a different configuration ofelectromagnets. For example, certain embodiments may comprise twoelectromagnets at the same level, but wired in opposite polarity so thatthe magnetic flux jumps the gap between the electromagnets (similar tothe spark in a spark plug). Other embodiments may compriseelectromagnets that alternate polarity along an array of adjacent PCRchambers, for the effect of concentrating flux in the zone of the PCRchamber. Certain embodiments may comprise electromagnets with variousback iron configurations to control the shape of the magnetic fluxfield.

It should be understood that the present devices and methods are notintended to be limited to the particular forms disclosed. Rather, theyare to cover all modifications, equivalents, and alternatives fallingwithin the scope of the claims. For example, in certain embodimentsdifferent configurations of magnets and or moveable magneticallyresponsive components may be used. In addition, other embodiments mayuse different time periods for holding shaft and moveable magneticallyresponsive components in the different positions.

The above specification and examples provide a complete description ofthe structure and use of an exemplary embodiment. Although certainembodiments have been described above with a certain degree ofparticularity, or with reference to one or more individual embodiments,those skilled in the art could make numerous alterations to thedisclosed embodiments without departing from the scope of thisinvention. As such, the illustrative embodiment of the present devicesis not intended to be limited to the particular forms disclosed. Rather,they include all modifications and alternatives falling within the scopeof the claims, and embodiments other than the one shown may include someor all of the features of the depicted embodiment. Further, whereappropriate, aspects of any of the examples described above may becombined with aspects of any of the other examples described to formfurther examples having comparable or different properties andaddressing the same or different problems. Similarly, it will beunderstood that the benefits and advantages described above may relateto one embodiment or may relate to several embodiments.

The claims are not to be interpreted as including means-plus- orstep-plus-function limitations, unless such a limitation is explicitlyrecited in a given claim using the phrase(s) “means for” or “step for,”respectively.

REFERENCES

The following references are incorporated herein by reference:

-   U.S. Pat. No. 5,352,036-   U.S. Pat. No. 6,176,609-   U.S. Pat. No. 6,357,907-   U.S. Pat. No. 5,578,201-   U.S. Pat. No. 8,048,375-   U.S. Pat. No. 8,052,929-   U.S. Pat. No. 8,550,694

We claim:
 1. A method of mixing reagents, the method comprising:obtaining an apparatus comprising: a chamber containing a magneticallyresponsive component and reagents; a heating module; an insertconfigured to (i) receive the chamber, and (ii) conduct thermal energyfrom the heating module to the chamber; and a magnet coupled to arotating shaft, where: the shaft is configured to move from a firstshaft position to a second shaft position; in the first shaft position,the second end of the magnet is distal from the chamber; and in thesecond shaft position, the second end of the magnet is proximal to thechamber; and moving the shaft from a first position to a secondposition, wherein the magnetically responsive component is moved withinthe chamber from a first position to a second position, thereby mixingthe reagents.
 2. The method of claim 1 wherein the chamber comprises abottom surface and a side surface, and wherein the magneticallyresponsive component contacts the bottom surface in the first positionand wherein the magnetically responsive component contacts the sidesurface in the second position.
 3. The method of claim 1 wherein: themagnetically responsive component is moved from the first position tothe second position and held in the second position for approximately 3seconds; and the magnetically responsive component is moved from thesecond position to the first position and held in the first position forapproximately 3 seconds.
 4. The method of claim 3 wherein themagnetically responsive component is cycled between the first and secondpositions for approximately 90 seconds.
 5. The method of claim 1 whereinat least one of the reagents is provided in a lyophilized form.
 6. Themethod of claim 1 wherein the side surface of the chamber is tapered andthe bottom surface of the chamber is curved.
 7. The method of claim 6wherein: the bottom surface is curved with a first radius; the moveablemagnetically responsive component is a spherical ball with a secondradius; and the first radius is greater than the second radius.
 8. Themethod of claim 1, comprising moving the magnetically responsivecomponent prior to beginning a polymerase chain reaction.
 9. The methodof claim 1, comprising moving the magnetically responsive componentduring at least a portion of a polymerase chain reaction.
 10. The methodof claim 9, wherein movement of the magnetically responsive componentoccurs during a temperature ramping phase.
 11. The method of claim 9,wherein movement of the magnetically responsive component from the firstposition to the second position reduces a temperature gradient in thechamber.
 12. The method of claim 1, comprising moving the magneticallyresponsive component prior to and during a polymerase chain reaction.13. The method of claim 1, comprising moving the magnetically responsivecomponent prior to beginning a reverse transcription reaction.
 14. Themethod of claim 1, comprising moving the magnetically responsivecomponent during at least a portion of a reverse transcription reaction.15. The method of claim 1, comprising moving the magnetically responsivecomponent prior to and during a polymerase chain reaction.
 16. A methodof mixing reagents, the method comprising: obtaining an apparatuscomprising: a chamber containing a magnetically responsive component andreagents; and a magnet coupled to a rotating shaft, where: the shaft isconfigured to move from a first shaft position to a second shaftposition; in the first shaft position, the second end of the magnet isdistal from the chamber; and in the second shaft position, the secondend of the magnet is proximal to the chamber; and moving the shaft froma first position to a second position, wherein the magneticallyresponsive component is moved within the chamber from a first positionto a second position, thereby mixing the reagents, wherein movement ofthe magnetically responsive component from the first position to thesecond position inverts a wax lyophilized layer in the chamber.
 17. Themethod of claim 1 wherein the magnetically responsive component is asphere.
 18. The method of claim 17 wherein the sphere has a diameter ofapproximately 0.0625 inches.
 19. The method of claim 1 wherein themagnetically responsive component is a disk or a sphere having a firstdiameter, and wherein a distance from first position to the secondposition of the magnetically responsive component is between two andfive times the first diameter.
 20. The method of claim 1 wherein thereagents are polymerase chain reaction (PCR) reagents.